Inflammatory diseases at both mucosal and non-mucosal sites are responsible for disease in millions of people worldwide. We found that a single oral dose of a common probiotic bacteria, Bacillus subtilis protects mice from intestinal inflammation caused by infection with an enteric pathogen, Citrobacter rodentium. B. subtilis is effective if administered as many as 3 days after C. rodentium, a time at which the pathogen has already induced early inflammatory processes. These findings suggest that B. subtilis may not only prevent inflammation, but more importantly, attenuate it. We have identified and purified a molecule, exopolysaccharide (EPS) from B. subtilis that when introduced by itself in a single intraperitoneal injection, prevents colitis induced by enteric pathogens. EPS binds F4/80+CD11b+ macrophages, and we have data indicating that protection by EPS is mediated by M2 macrophages in a TLR4-dependent manner. Further, we found that a 2nd (TLR2-dependent) cell type is required for protection by EPS. We hypothesize that EPS induces the generation of M2 macrophages which secrete cytokines that either render inflammatory T cells non-responsive, or induce development of regulatory T cells. Here, we will establish if M2 macrophages mediate protection and if EPS stimulates these cells via TLR4 signaling. Further, we will test if M2 macrophages secrete anti-inflammatory cytokines and if they inactivate T cells or induce regulatory T cells. Finally, we will use three disease models (food allergy, autoimmunity and inflammatory bowel disease) to test if EPS is effective in acute and/or chronic inflammatory diseases. We have preliminary data indicating that B. subtilis and EPS can protect from food allergy which affects millions of people. Experiments proposed in this grant will enhance our molecular understanding of how probiotic molecules function to treat and/or prevent disease, and lead to the rational application of small therapeutic probiotic molecules to humans.